1. Field of the Invention
This invention relates to methods for isolation of rare proteins from bacterial samples. More particularly, this invention relates to a method for isolating rare outer membrane proteins from the family Spirochaetaceae, such as genus Treponema and to the use of such proteins in diagnosis and prophylaxis of related diseases.
2. Description of Related Art
The genus Treponema (order Spirochaetales, family Spirochaetaceae), a type of gram-negative bacteria, contains four human pathogens as well as at least six nonpathogens. The pathogens are characterized by an extreme sensitivity to environmental conditions that renders them impossible to culture in vitro. Due to DNA homology the agents that cause syphilis, yaws and endemic syphilis have been combined into one species and three subspecies: T. pallidum subsp. pallidum (syphilis); T. pallidum subsp. pertenue (yaws); and T. pallidum subsp. endemicum (endemic syphilis). T. carateum, which is the causative agent of pinta, remains a separate species. Syphilis is found worldwide, yaws is endemic in the tropics, pinta is prevalent in tropical areas of Central and South America, and endemic syphilis is restricted to desert regions. These treponemal infections are very complex, each exhibiting distinct stages of symptomatic manifestations followed by asymptomatic periods. Without antibiotic therapy, these diseases are chronic and may last for 30 to 40 years.
To date, the four pathogens have been considered antigenically identical. An individual subspecies-specific antigen has not been identified and serological reactions demonstrate immunological relatedness. Both Wassermann and anti-T. pallidum subsp. pallidum antibodies develop in response to each treponemal disease, and known protective immunogens are also related, as shown by cross-resistance (T. B. Turner, et al., Biology of the Treponematoses. W.H.O Monogr. Ser. 35:1-277, 1957). Therefore, the geographical location together with the clinical manifestations of the patient have been considered the key to diagnosis (Manual of Clinical Microbiology, 5th Ed., A. Balows, et al., Eds., p 567, 1991).
Freeze-fracture electron microscopy of outer membranes from pathogenic spirochetes has revealed that their integral transmembrane outer membrane protein density is one to two orders of magnitude less than that of typical gram negative bacterial pathogens. It has been proposed that this low outer membrane composition, and thus low surface exposure of antigenic target molecules, allows these organisms to effectively evade the host immune response, contributing to the chronic nature of infection exhibited by all spirochetal pathogens.
As is well known, the outer membranes of spirochetes, including that of Treponema pallidum subsp. pallidum, the agent of syphilis, are fragile structures as compared to those of typical gram negative bacteria. Consequently, separation of the outer membrane from the inner membrane has proven extremely difficult. Certain other medically relevant spirochetal bacteria with outer membrane structure and, hence, protein structure, similar to those of T. pallidum include Borrelia burgdorferi (Lyme Disease), Borrelial species (relapsing fever), and Leptospiral species (leptospirosis).
The outer membrane of T. pallidum has been found to be antigenically inert and resistant to specific treponemidical antibody (Radolf, et al., Infect. Immun., 52:579, 1986; Hovind-Hougen, et al., Acta Pathol. Microbiol. Scand., 87:263, 1979; Nelson, et al., J. Exp. Med., 89:369, 1949). Yet freeze-fracture electron microscopy has shown that certain rare outer membrane protein (tromp) molecules of T. pallidum have surface exposed antigenic sites that bind antibody present in the serum of challenge immune animals (Blanco, et al., J. Immunol., 14:1914-1921, 1990). Taken together the spirochetal bacterial pathogens imperil the health of a considerable portion of the human population, yet development of effective and specific vaccines and isolation of antigens capable of generating protective immune response has been hampered by a considerable number of problems associated with this organism: the impossibility of culturing the T. pallidum in vitro, the limited numbers of organisms that can be obtained from infected animals, the contamination of treponemes by host tissue components, the fragility of the treponemal outer membrane, and the difficulty of isolating and identifying the outer membrane proteins of pathogenic spirochetes.
Previous studies attempting to identify transmembrane outer membrane proteins of pathogenic spirochetes have utilized various detergent solubilization approaches. Spirochetal outer membranes bleb form the underlying protoplasmic cylinder under relatively mild conditions, including dilute detergents and hypotonic environments. However, such apporaches have identified only abundant subsurface located proteins, including various lipoproteins which by definition are not transmembrane molecules and do not form particles viewed by freeze-fracture analysis.
Therefore, the need exists for new and better vaccines based upon the identification of virulence related outer membrane molecules to be used in diagnosis and for prophylaxis of diseases related to the pathogenic spirochetal bacteria, especially the genus T. pallidum. 
A novel method is provided for isolating outer membrane of pathogenic Spirochaetacae family without use of detergents. The pathogen is purified from contaminating host components using a density gradient centrifugation medium that is stable at pH from about 3.2 to about 3.0, preferably a Ficoll step gradient. The purified pathogen is treated with a lipid soluble chromophore that intercalates into outer membrane to provide a visual marker of membrane matter. Outer membrane is released from protoplasmic cylinders using a hypotonic, low pH buffer, preferably citrate, followed by density gradient centrifugation, which yields, for example, the chromophore labeled bands at 7% and 35% sucrose (wt/vol) for T. pallidum and T. vincentii, respectively.
Freeze-fracture electron microscopy of membrane vesicles from the spirochete reveals an extremely low density of protein particles. For instance, the particle density of T. pallidum is approximately six times less than that of T. vincentii. Comparative immunoblot analysis of the T. vincentii membrane material to that of whole organisms showed a lipopolysaccharide (LPS) ladder consistent with 20% recovery of the outer membrane. Immunoblots of T. vincentii outer membrane also showed two antigenic proteins at 55- and 65-kDa.
125I-penicillin, which binds only to inner membrane and not to outer membrane, was used to detect the presence of any inner membrane in the outer membrane preparation isolated according to the method of the invention. No penicillin binding proteins in the T. pallidum membrane material were detected, indicating the absence of inner membrane contamination.
Immunoblot analysis of T. pallidum outer membrane using antibodies specific for periplasmic associated proteins showed no detection of known 19-kDa xe2x80x9c4Dxe2x80x9d protein or the 47-kDa lipoprotein and only trace amounts of endoflagellar protein. Rare outer membrane proteins associated with the T. pallidum were detected by one and two dimensional reducing SDS-PAGE separation and immunoblot analysis, using gold staining and serum from infected and challenge immune animals. As compared to whole organism preparations, four of the isolated proteins were obtained in significantly enriched amounts from the outer membrane preparation.
Methods are provided for the use of outer membrane proteins of pathogenic Spirochaetacae family for detection and amelioration of associated disease states.
The isolation of the T. pallidum outer membrane and identification of its protein constituents has been complicated by the fragility of this structure, the limited number of treponemes that can be acquired by rabbit infection, and the significant amount of host contaminating protein following extraction of organisms from infected animals. Moreover, freeze-fracture electron microscopy has revealed that the outer membrane of T. pallidum contains two orders of magnitude less integral membrane protein than typical gram negative bacteria (Radolf, et al., Proc. Natl. Acad. Sci. USA, 86:2051-205-5,1989; Walker, et al., J. Bacteriol., 171:5005-5011, 1989). Because of the paucity of T. pallidum rare outer membrane protein (TROMP), it is likely that previous studies using detergent extraction of T. pallidum to identify transmembrane outer membrane proteins have mistakenly identified as outer membrane proteins abundant subsurface molecules, including lipoproteins anchored in the inner membrane that are released by such treatments (Chamberlain, et al., Infect. Immun., 57:2872-2877, 1989; Penn, et al., Immunology, 46:9-16, 1982; Penn, et al., J. Gen. Microbiol., 131:2349-2357, 1985).
It has previously been shown that while 0.1% Triton X-114 can selectively solubilize the T. pallidum outer membrane, some subsurface molecules, including the 47-kDa lipoprotein, are also released (Radolf, et al., Infect. Immun., 56:490-498, 1988). Concentrations of Triton X-114 of up to 2% have been shown to release additional T. pallidum lipoproteins (Cunningham, et al., J. Bacteriol., 1 70:5789-5796, 1988; Radolf, et al., supra, 1988).
The present invention provides a method for isolating the outer membranes from treponemes and other spirochetes with rare outer membrane proteins in the absence of detergents. In the examples herein this procedure was applied to T. vincentii, which, because of the LPS content of its outer membrane, was used as a marker for outer membrane recovery. Preliminary studies showed that while a hypotonic osmotic environment caused significant blebbing of the treponemal outer membrane, only a small amount of outer membrane was released. Endoflagellar filaments may physically interact with the outer membrane in the process of motility (Berg, J. Theor. Biol., 56:269-273, 1976; Goldstein, et al., Cell Motility and the Cytoskeleton, 9:101-110, 1988). These structures may limit the release of outer membrane under hypotonic conditions. Therefore, in the present invention a low pH hypotonic buffer is used to dissociate endoflagellar filaments (Blanco, et al., Infect. Immun., 56:168-175, 1288). As a result, the cuter membrane is completely released as viewed by electron microscopy. The low pH treatment, however, is incompatible with purification of T. pallidum by the conventional Percoll procedure due to the adverse effects of low pH on residual Percoll, which solubilizes in low pH conditions. Therefore, in the practice of this invention, T. pallidum is purified using a continuous or discontinuous density gradient separation in a medium that is stable in the pH range from 3.2 to 3.0, removes contaminating host components, and is also compatible with the subsequent low pH incubation.
A second key step in the practice of this invention is treatment of treponemes with a chromophore, preferably one that intercalates into biological and liposomal membranes. The preferred chromophore is octyl-decyl rhodamine, but one skilled in the art will appreciate that any chromophore of a size suited to intercalate into liposomal membranes can be used so long as it is naturally lipophilic or can be substituted with lipid-solubilizing moieties containing between 8 and 10 carbon atoms. In addition, the lipid-soluble chromophore should be selected so as not to significantly alter the membrane particle density. Use of the chromophore provides a visual marker to follow the disposition of released outer membrane. To determine whether the chosen lipid-soluble chromophore alters membrane particle density, a pathogenic spirochete having an order of magnitude greater amount of outer membrane protein than the one being isolated can be used. For instance, using Borrelia burgdorferi, a pathogenic spirochete which has an order of magnitude greater amount of outer membrane protein than T. pallidum (Walker, et al., supra, 1991), it was found that octyl-decyl rhodamine did not change its outer membrane particle density (data not shown), suggesting that the outer membrane proteins of T. pallidum and T. vencentii were also not affected by this reagent.
The finding herein that T. pallidum outer membrane banded in a sucrose gradient at a very low density (7%) is consistent with membrane that contains a small amount of protein (Tomlinson, et al., Biochem., 28:8303-8311, 1989). This finding was further confirmed by freeze-fracture electron microscopy of purified T. pallidum membrane vesicles, which showed fracture faces that contained extremely rare intramembranous particles. This result is similar to the low particle density observed by others for the native outer membrane of T. pallidum (Radolf, et al. supra; Walker, et al., supra). By comparison, the T. vincentii outer membrane banded in a sucrose gradient at a higher density (35%) as is consistent with the greater amount of intramembranous particles observed in its membrane and/or is consistent with a membrane that contains lipopolysaccharide (LPS).
The selective isolation of the T. pallidum outer membrane from the protoplasmic cylinder was determined by the use of penicillin binding proteins (PBPs) as a marker to visualize inner membrane associated proteins. Previously studies have shown that T. pallidum PBPs remain with the protoplasmic cylinders following solubilization of the outer membrane in the detergents Triton X-114 or Triton X-100 (Cunningham, et al., J. Bacteriol., 169:5298-5300, 1987; Radolf, et al., Infect. Immun., 57:1248-1254, 1989). No PBPs were detected with purified outer membrane prepared according to the method of this invention, indicating that the procedure selectively removes only the outer membrane, free from contamination by inner membrane.
Of particular significance is the complete absence of the T. pallidum outer membrane preparation so the 4D protein and the 47-kDa major lipoprotein, and the finding of only trace amounts of endoflagellar protein, indicating little to no contamination by these periplasmic components. The 47-kDa lipoprotein, one of the most abundant T. pallidum molecules, was not detected in the outer membrane preparation, thus confirming that inner membrane anchored lipoproteins were not released by this procedure.
Coomassie stained SDS-PAGE and immunoblot analysis of 1xc3x97109 T. vincentii equivalents of outer membrane revealed two major antigenic protein species of 65- and 55-kDa. In contrast, Coomassie stained SDS-PAGE of a 5-fold greater amount of T. pallidum outer membrane showed no detectable protein. These findings are consistent with the observations of freeze-fracture electron microscopy indicating that the outer membrane particle density of T. pallidum is six times less than that of T. vincentii. From the outer membrane particle density of T. pallidum, which has been determined to be 170 particles/um2 and the surface area of T. pallidum, which is approximately 4 um2, it is calculated that 5xc3x97109 T. pallidum should contain only 250 nano grams of outer membrane protein based upon a single species of 50K molecular weight.
Therefore, the amount of a single species of TROMP isolated using the method of this invention is several hundred times less than was previously erroneously identified by prior art methods (Norris, et al., Microbiol., 57:750-779, 1993).
Enhanced chemiluminescence (ECL) immunoblotting has the sensitivity of detecting pico grams of antigen (ECL Western Blotting protocols, Buckinghamshire, England, 1993). Therefore, this technique is preferably employed in the method of this invention for detecting outer membrane associated protein. Most preferably, using ECL, immunoblots of outer membrane samples are prepared in urea and electrophoresed in one dimension or following two dimensional (2D) electrophoresis, and probed with sera from rabbits with immunity to the pathogenic treponeme of interest. Using this technique upon T. pallidum showed two major antigenic protein bands at 17- and 45-kDa. The 17-kDa protein had a pI of greater than 7.0, showed higher oligomeric forms, and selectively partitioned into the hydrophobic phase following Triton X-114 detergent extraction (data not shown).
These findings are consistent with the properties of the native and recombinant 17-kDa lipoprotein of T. pallidum (Atkins, et al., Infect. Immun., 61:1202-1210, 1993). It was also shown using specific monoclonal antibodies that the 45-kDa protein was the previously characterized TmpA lipoprotein (Schouls, et al., Microb. Pathog., 7:175-188, 1989; Hansen, et al., J. Bacteriol., 162:1227-1237, 1985). While the vast majority of these two proteins remain associated with the protoplasmic cylinder following outer membrane removal (data not shown), some of the 17- and 45-kDa lipoproteins are specifically associated with outer membrane.
In addition to the strongly antigenic 17- and 45-kDa lipoproteins of T. pallidum isolated, gold-stained 2D blots of 3xc3x971010 treponemal equivalents revealed four additional T. pallidum proteins, including one each at 28- and 65 kDa, and two at 31-kDa. All of these proteins have been found to contain antigenic sites reactive with sera from immune rabbits. Comparison of the pI""s of these found proteins to those on 2D blots of 5xc3x97108 whole organisms have shown that the 31-kDa (acidic pI) and 28-kDa proteins correspond to prominent T. pallidum protein spots on immunoblots and may be additional outer membrane associated lipoproteins not heretofore identified. In contrast, the 31-kDa protein (basic form) corresponds to a minor and faintly detectable protein spot on 2D blots of whole organisms, while the 65-kDa protein does not correspond to any previously identified T. pallidum protein. In view of their significant enrichment following outer membrane isolation, the 31-kDa (basic pI) and 65-kDa proteins are identified as rare outer membrane proteins.
The outer membrane proteins of typical gram negative bacteria include an export signal cleaved by leader peptidase I, and amphiphathic beta pleated sheet structure throughout the secondary sequence that generates membrane spanning regions (Vogel,et al., J. Mol. Biol., 190:191-199, 1986; Weiss, et al., Science, 254:1627-1630, 1991; Von Heijne, J. Mol. Biol., 184:99-105, 1985). Recently, the gene encoding a surface exposed 31-kDa protein of Leptospira alstoni, designated Omp-LI (outer membrane protein of Leptospira), has been cloned, sequenced, and expressed (Haake, et al., J. Bacteriol., 175:42254234, 1993). The deduced amino acid sequence of this protein shows an export and amphiphatic beta pleated sheet topology resulting in 10 membrane spanning domains (Haake, et al., supra). The structural similarity between this putative outer membrane protein of Leptospira and those of typical gram negative bacteria suggests that other spirochetal outer membrane proteins may be structurally similar to those of typical gram negative bacteria. The Leptospira outer membrane has been isolated using the method of this invention. The membrane material purified was found to selectively contain lipopolysaccharide like substance (LLS), which is unique to the Leptospira outer membrane (Zeigler, et al., Can. J. Microbiol., 21:1102-1112, 1975), and several proteins including OMP-LI. These findings provide additional evidence that the membrane material and associated protein isolated by the method of this invention from T. pallidum is outer membrane in origin.
The binding of antibody in immune serum to virulent T. pallidum results in aggregation of TROMP particle as viewed by freeze fracture electron microscopy (Blanco, et al., supra, 1990). These findings have recently been confirmed and extended using serum obtained from animals with varying degrees of challenge immunity. Particle aggregation directly correlates with the development of challenge immunity, suggesting that TROMP are key targets for a protective host immune response.
In addition, due to isolation and purification of the T. pallidum outer membrane, the amino acid sequence of the protein has been obtained and of DNA encoding it and for cloning of TROMP molecules. The recombinant expression of these rare outer membrane proteins can be used for experimental biology studies to address directly the molecular basis for T. pallidum pathogenesis, for diagnostic tests to detect syphilis and for development of host immunity during syphilis.